Ewing's sarcoma (ES) is a solid highly malignant neoplasm of the bone and soft tissues. Most often it affects children and young adolescents, being the second most common malignant bone tumor in young adults. ES is formed by poorly differentiated round cells of neuroectodermal origin. Current ES treatment includes a combined modality with radiotherapy and chemotherapy. Clinically, ES tumors are generally responsive to such treatment, but the overall cure rate is low because ES is an aggressive osteolytic tumor that frequently presents with metastatic disease. ES cells are characterized for having, in nearly 100 percent of the cases a reciprocal translocation between chromosomes 11 and 22 or, much less frequently, 21 and 22. This translocation results in the synthesis in these cells of fusion proteins with their N-terminus encoded by EWS gene sequences and their C- terminus encoded by sequences of either the FLI-1 gene, most frequently, or the ERG gene, both members of the ETS family of transcription factors. There is a limited repertoire of fusion types, with those involving EWS/FLI-1 being most frequently detected in ES patients. We have found that down-regulation of EWS/FLI-1 in ES cells caused both growth inhibition and sensitization to apoptosis by ionizing radiation and chemotherapeutic agents. Our overall objective is to define antisense oligonucleotides targeted to the EWS/FLI-1 (or EWS/ERG) junction as highly specific therapeutic tools for ES management, because there are no target fusion sequences in normal cells. Our central hypothesis is that antisense EWS/FLI-1 oligonucleotides targeted to the translocation junction will specifically render ES cells sensitive to DNA-damaging agents and, in addition, will prevent the oncogenic activity of the EWS/FLI-1 protein product. Treatment with antisense EWS/FLI-1 oligonucleotides will have a dual effect, enhancing ES cell killing and down-regulating cellular neoplastic properties. Two specific aims are proposed, which include experiments designed to study the effects of antisense EWS/FLI-1 oligonucleotides in vitro, on cultures ES cell lines, and in vivo, on tumors induced in mice by injection of ES cells. These studies should advance technological and scientific understanding for the translational application of antisense EWS/FLI-1 oligonucleotides to ES treatment in the future.